A. Specific Aims Our goal is to better understand the principles that underlie protein folding and structure. The vast increase of our knowledge of genomes needs to be matched by a corresponding increase in our understanding of the fundamental links between sequences and folded structures, and the ability to predict protein conformations. This collaborative project will involve the efforts of Drs. Wesley Stites (protein mutagenesis and thermodynamic characterization; protein crystallography), Charles Wilkins (mass spectroscopy), Peter Pulay (computational chemistry), James Hinton (protein NMR) specific aims will be: 1. To examine critically the importance of hydrogen bonding and hydrogen bond networks for protein folding and conformational stability, including (enhanced) thermal stability. Both side-chain and main- chain/side-chain interactions will be examined using carefully selected designed mutations, under conditions where we will attempt to maintain nearly equivalent packaging and van der Waals interactions. The hypothesis that thermophilic organisms achieve stability (in part) through networks of hydrogen bonds will be tested by comparing, extending and building upon existing hydrogen bond interactions in mesophilic vis-a-vis thermophilic proteins. 2. To examine the critically the importance of hydrophobic packing interactions for protein folding and conformational stability, using a (very) extensive set of isoleucine, leucine and valine substitutions. in the major hydrophobic core of staphylococcal nuclease. 3.To establish correlations between folding energetics and protein three- dimensional structures.. This goal will be achieved examining at high resolution the structures of the mutant proteins whose folding energetic are determined in aims 1 and 2, above. Structural analysis will be accomplished by X-ray crystallography to a resolution <2.0 A, and (for selected mutants) by NMR spectroscopy in solution. 4. To develop a new method for the prediction of the structural and energetic effects of packing mutations in proteins. The method will use efficient internal coordinate optimization for calculating the structures and energetics of native (folded) proteins, and for molecular dynamics simulations of unfolded states. The approach will take advantage of the emerging database of structural and energetic factors that we will establish in aims 1-3 and will develop enhanced predictive methods.